Endotoxins of gram-negative bacteria were found to react with the lysate of L. polyphemus amebocytes to form fine precipitin-like particles in the presence of large quantities of pyrogen-free sterile water. Light scatter measurements during the formation of these particles indicated that the rate of formation to be a function of endotoxin concentration. A nephelometric procedure for the quantitation of pyrogens (endotoxins of gram-negative bacteria) with Limulus amebocyte lysate (LAL) was developed and optimized by studying the various parameters of the reaction. The finalized procedure was established as 200 microliters of LAL reagent with 100 microliters of an endotoxin solution (reference or unknown solution) diluted to 2 ml with pyrogen-free sterile water. The end-point of the analysis was chosen as that time (minutes) at which the light scattering capacity of the particles reached 10 light scatter units above is baseline value. Studies indicated that the end-point data obtained on different levels of endotoxin can be defined with a mathematical formula. The standard curve data plotted on semi-log graph paper was a straight line. Reproducibility studies showed that the method was capable of giving a provision of 13% when testing endotoxin at a level of 100 pg/ml. The procedure permitted the quantitation of endotoxin as low as 16 pg/ml.